Regulation of the Expression of the Hematopoietic Stem Cell Antigen CD34: Role of C-Myb by Paola Melotti, De-Hui Ku, and Bruno Calabretta

Brief Det~nitive Report

Regulation of the Expression of the Hematopoietic Stem Cell Antigen CD34: Role of c-myb By Paola Melotti, De-Hui Ku, and Bruno Calabretta

From the Department of Microbiolagy and Immunology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Summary The CD34 antigen defines a subset of hematopoietic progenitor cells with self-renewal capacity and the ability to reconstitute hematopoiesis in irradiated primates and marrow-ablated humans, but its function remains unknown. The c-myb protooncogene plays a fundamental role in hematopoiesis, most likely via its transcriptional regulator function. We report that c-myb protein Downloaded from http://rupress.org/jem/article-pdf/179/3/1023/1104540/1023.pdf by guest on 02 October 2021 transactivates the CD34 promoter via specific interaction with multiple Myb binding sites in the 5' flanking region of the gene and induces expression of the endogenous CD34 mtLNA in rodent fibroblasts. Also, constitutive expression of c-myb in CD34-negative human glioblastoma cells induces expression of CD34 mRNA and synthesis of the surface membrane antigen. These data directly demonstrate that c-myb regulates the expression of the hematopoietic stem cell antigen CD34 and raise the possibility that c-myb regulates hematopoiesis inducing a cascade of differentiation-related events.

he CD34 surface antigen is a highly glycosylated trans- Materials and Methods T membrane protein expressed on hematopoietic stem cells Gel RetardationAssay. HB101cells containing the parental pFlag and lineage-specific progenitor cells, on a subset of bone (IBI, New Haven, CT) expression vector only or HB101 cells con- marrow stromal cells and on small vessel endothelium of a taining the pc-Myb Flag vector (20) were incubated for 4 h in the variety of tissues (1-4). About 1-4% of normal bone marrow presence of 1.5 mM isopropyl/~-D-thiogalactopyranoside (IPTG) cells and ",,30% of blasts from acute leukemia patients ex- to an OD 600 of 0.500. Myb expression was determined in bac- press CD34 (1, 2, 5). To date, CD34 remains the only well- terial lysates by Western blot using an anti-Myb-specific antibody defined human stem cell marker. CD34-positive cells have (Upstate Biotechnology Incorporated [UBI], Lake Placid, NY). self-renewal capacity and ability to reconstitute hematopoiesis 32p-labeled double-stranded probes were synthesized by PCR and in sublethally irradiated primates and marrow-ablated humans corresponded to three different segments of the human CD34 5' flanking region from -294 to -115; -132 to 54, and 67 to 234, (6, 7). The function of CD34 is not yet known, although based on the published sequence (9, 11). Gel retardation assays were this protein has been proposed to play a role in hematopoietic performed as described (20). cell adhesion to stromal cells, perhaps facilitating the interac- TransientChloramt~henicol Acetyl-Transferase (CAT) Analysis. Con- tion with locally released growth factors, and to directly func- structs in which different CD34 promoter regions drive the bac- tion as a signal transducer (3, 4, 8). terial CAT gene were prepared by PCR amplification of human The mechanisms regulating CD34 expression in hemato- placental genomic DNA and by cloning the different fragments poietic cells are not well understood, although there is evi- of the CD34 5' flanking region first into the pCRII vector (In- dence of transcriptional and posttranscriptional regulation of vitrogen Corp., San Diego, CA) or directly into the pUCCAT vector CD34 expression (9, 10). In light of putative Myb binding (Promega Corp., Madison, WI). CD34 SM-CAT and CD34 LM- sites present in the 5' flanking region of the CD34 gene (9, CAT contain identical nucleotide substitutions in the Myb consen- 11), and the fundamental role of c-myb in hematopoietic cell sus sequence, from nucleotide 75 to 78 and 92 to 95 (CAAC to TGGC and GTTA to GCCC, respectively), of the published se- proliferation and/or differentiation (12-15), most likely through quence of the human CD34 gene (9, 11). TK-ts13 hamster fibro- the transcription regulator function of Myb proteins (16-19), blasts were transfected, using the calcium-phosphate precipitation we investigated the possibility that c-myb is directly involved method (21), with 1 #g of CAT reporter plasmid with or without in transcriptional regulation of CD34 gene, possibly by in- 5 #g of effector plasmid (pMbl-dhfr, named pSV myb, containing ducing expression of its mRNA and synthesis of the surface the human c-myb cDNA driven by SV40 early promoter) plus 1 membrane protein in nonexpressing cells. /zg of a plasmid containing the bacterial fl-galactosidase gene driven

1023 J. Exp. Med. The Rockefeller University Press ~ 0022-1007/94/03/1023/06 $2.00 Volume 179 March 1994 1023-1028 by the DNA polymerase-c~promoter, as an internal control oftrans- fection efficiency.Cells were harvested 48 h after transfection. Pro- teins were extracted by freeze-thawing and normalized for transfection efficiencyusing the 3-galactosidase assay, as suggested by the manufacturer (Promega). Cellular lysates were incubated with [14C]chloramphenicol and acetyl-CoA for 1 h at 37~ Transacti- vation of the reporter constructs was assayed by measuring the amount of acetylated [14C]chloramphenicolby thin-layer chroma- tography followed by autoradiography and scintillation counting. Detection of c-myb Protein in Transfected Cells. Levels of c-myb Figure 1. Myb protein binding to CD34 5' flankingregion. Lanes 1, protein were determined in total cell extracts from 6 x 10 6 trans- 4, 7, and I0, free probe only; lanes 3, 6, 9, and 12, probe plus 1/xg of fected TK-ts13 hamster fibroblasts and human glioblastoma T98G parental bacterial lysate;lanes 2, 5, 8, and 11, probe plus 1 #g of bacterial cells by Western blot analysiswith a monoclonal anti-mouse c-myb lysatecontaining the humanc-myb protein. The probe used in lanes 10-12 antibody (UBI) and then with a peroxidaselabeled sheep anti-mouse contains three nucleotide substitutions in both Myb binding sites. The Ig antibody (Amersham Corp., Arlington Heights, IL). Bound different regions of the CD34 promoter used as probe are indicated on antibodies were revealed with the Enhance Chemiluminescence the top. Detection System (ECL; Amersham Corp.). Expression of CD34 mRNA in Transfected Cells. TKots13cells were transfected with no plasmid, 5 #g of pSV 3-gal containing Downloaded from http://rupress.org/jem/article-pdf/179/3/1023/1104540/1023.pdf by guest on 02 October 2021 bacterial 3-galactosidase DNA under the control of the SV40 early consensus sequences was used (Fig. 1, lane tt), demonstrating promoter or 5/xg of pSVmyb. RNA was extracted as described (22) 24, 36, or 48 h after transfection. Using 0.7/~g of total RNA the specificity of the interactions. and 40 cycles CD34 mRNA was amplifiedby reverse transcriptase CD34 Promoter Activity Is 7~ansactivated by c-myh To deter- (RT)-PCR, as described (23), with a pair of synthetic primers cor- mine whether c-myb transactivates the CD34 5' flanking re- responding to nucleotides from 461 to 482 (5' primer) and from gion, constructs in which different fragments of the CD34 246 to 267 (3' primer) of the published routine CD34 cDNA se- promoter drive bacterial CAT gene expression were prepared quence (24). AmplifiedDNA was subjected to electrophoresis, trans- by cloning the segments corresponding to nucleotides -666 ferred to Zetabind nylon filters (Cuno, Inc., Meriden, CT) and de- to 234 (CD34 L-CAT), -132 to 234 (CD34 M-CAT), and tected by Southern hybridization with a y-[32p]ATP end-labeled 31 to 187 (CD34 S-CAT) (9, 11) into the pUCCAT vector oligoprobe corresponding to nucleotides 366 to 395 (24). In TK- (Fig. 2 A), and transfected into wild-type TK-ts13 Syrian tsD cells constitutively expressing c-myb, CD34 mR.NA levels were hamster fibroblasts or in cells constitutively expressing c-myb also measuredby RT-PCR using 0.7/zg of total RNA and 40 cycles. In the different T98G cell lines CD34 mRNA levels were also de- (SV-mybTK-ts13). TK-ts13 cells were transfected at a 5:1 termined by RT-PCR. Primers used correspond to nucleotides 957 effector-to-reporter ratio with plasmid pMbm-dhfr. This plas- to 982 (5' primer) and 1165 to 1190 (3' primer); the oligoprobe mid, which contains the human c-myb cDNA under the con- includes nucleotides 1109 to 1134, of the published human cDNA trol of the SV40 early promoter and enhancer linked to the sequence (25). Amplification products were run on 1% agarose dehydrofolate reductase coding sequence for methotrexate se- gel, blotted and probed with the 32p end-labeled oligoprobe. lection (11) and the described above reporter plasmids, were CelISu~ace CD34 Expression. Exponentiallygrowing cells were used in the experiments. Levels of CAT activity were assayed harvested and incubated (30 min on ice in PBS containing 0.1% 48 h later (Fig. 2 B). c-myb induced 8-, 10-, and 14-fold in- gelatin, 0.01% sodium azide, 5 % fetal calf serum) with antibodies creases, respectively, in CAT expression driven by the CD34 to CD34 (mouse IgG1 anti-HPCA1; Becton Dickinson & Co., L-CAT (Fig. 2 B, lane 3), CD34 M-CAT (Fig. 2 B, lane 5), Mountain View, CA), 32-microglobulin (BBM1) as positive con- and CD34 S-CAT (Fig. 2 B, lane 7) CD34 5' flanking region trols, or CD16 (3G8, irrelevant IgG1), as negative controls. Cells were washed and incubated (30 min on ice) with FITC-conjugated segments. Transactivation was abolished when mutations were goat anti-mouse Ig F(ab')2. Cells were washed and analyzed by introduced in the Myb binding sites of the CD34 S-CAT flow-cytometry on a EPICS Profile Analyzer (Coulter Corp., Hia- reporter vector (CD34 SM-CAT; Fig. 2 B, lane 9). leah, FL). To determine whether the Myb binding sites not included in the segment corresponding to CD34 S-CAT (nucleotides 31 to 187) are also involved in CD34 5' flanking region trans- Results activation, TK-ts13 and SVmybTK-ts13 cells were transfected c-myb Protein Interacts with the 5' Flanking Region of the CD34 with CD34 LM-CAT containing the 900-bp fragment of the Gene. To determine whether Myb interacts with putative CD34 5' flanking region with mutations in the two most prox- Myb binding sites in the 5' flanking region of the CD34 gene, imal Myb binding sites (9, 11). Transactivation was unaffected gel retardation assays were performed with bacterial lysates (Fig. 2 B, lane 4), compared with the wild-type L-CAT con- containing or not containing Myb protein, and probed with struct (Fig. 2 C, bar 2), indicating that the more distal Myb different 32p-labeled DNA fragments of the CD34 5' flank- consensus sequences are also functional binding sites. CAT ing region (Fig. 1). One retarded complex was detected in assays were also performed using TK-ts13 or SVmybTK-ts13 the lysate containing Myb protein (Fig. 1, lanes 2, 5, and cells transfected with CD34 L-CAT and CD34 LM-CAT in 8), but none in the lysate that lacked Myb protein (Fig. 1, the presence of an excess (100:1, molar ratio) of a 36-bp frag- lanes 3, 6, 9, and 12). No binding was detected when a probe ment of the CD34 5' flanking region (nucleotides 31 to 67) with a 3-base substitution in each of the two potential Myb containing two wild-type or two mutated Myb binding sites.

1024 Role of c-myb in HematopoieticStem Cell Antigen CD34 Expression of CAT activity were identical in TK-ts13 cells transfected with pSVCAT in the presence or absence of an excess (100:1 molar ratio) of competitor (not shown). CD34 Expression Is Regulated by c-myt~ To test the predic- tion, from the above results, that c-myb may upregulate CD34 expression, RT-PCR analysis of CD34 mRNA expression was performed in TK-ts13 cells 24, 36, and 48 h after transfection with pSVmyb. CD34 mRNA was detectable only in c-myb-expressing cells (Fig. 3 A, lanes 3-5), but not in mock- transfected TK-ts13 cells (Fig. 3 A, lane 1), or in TK-ts13 cells transfected with the pSV3-gat plasmid (Fig. 3 A, lane 2) used as controls. In addition, CD34 mRNA expression was detected in SVmyb TK-ts13 cells constitutively expressing c-myb protein (Fig. 3 B, right, lane 2). Antibodies are available for detection of human, but not rodent CD34 proteins; therefore, to prove that induced CD34

mRNA expression results in synthesis and expression of the Downloaded from http://rupress.org/jem/article-pdf/179/3/1023/1104540/1023.pdf by guest on 02 October 2021 encoded protein, human glioblastoma T98G cells (nonexpressing c-myb) were transfected with a c-myb cDNA and assayed for Myb protein and CD34 expression. Western blot analysis revealed the presence of a 75-kD protein corresponding to c-myb, in SVmyb T98G cells (Fig. 4 A, lane 2), in SVmybM T98G cells selected with increasing concentrations of methotrexate (final concentration 22 #M) to induce amplification

Figure 2. c-mybtransactivation of CD34 5' flanking region in Tk-ts13 hamster fibroblasts. (A) Constructs in which different CD34 promoter regions drive the bacterial CAT gene. (B) Autoradiogramsof CAT activity in lysates of TK-ts13 transfectedwith the negative control puCCAT (lane 1), CD34 DCAT (lane 2), CD34 M-CAT (lane 4), CD34 S-CAT (lane 6), CD34 SM-CAT (lane 8), or cotransfected with pSV-myb (lanes 3, 5, 7, and 9, respectively).(C') Scintillation countings of acetylated [14C]chloramphenicol in lysates of: TK-ts13 (lane I), and SVmyb-TK-ts13cells (lane 2) transfected with CD34 I~CAT; TK-ts13 (lane 3) and SV-Myb TK-ts13 cells (lane 4) transfectedwith CD34 LM-CAT, TK-ts13(lane 5), and SVmyb TK-ts13 cells (lane 6) transfectedwith CD34 L-CATplus a synthetic competitor containing two wild-type Myb binding sites; TK-ts13 (lane 7) and SVmyb TK-ts13 cells (lane 8) transfectedwith CD34 LM-CAT plus a synthetic competitor containing two wild-type Myb binding sites; TK-ts13 (lane 9) and SVmyb TK-ts13 cells (lane 10) transfectedwith CD34 L-CAT plus a synthetic competitor containing two mutated Myb binding sites; Figure 3. CD34 mRNA levels in TK-ts13 cells transfectedwith pSV- and TK-ts13 (lane 11) and SVmyb TK-ts13 cells (lane 12) transfectedwith myb. (A) Expression of CD34 mRNA in transiently transfected TK-ts13 CD34 LM-CAT plus a synthetic competitor containing two mutated Myb cells. (Lane 1) cells transfected with no plasmid; (lane 2) cells transfected binding sites. with 5/zg ofpSV 3-gal; (lanes 3-5) cells transfectedwith 5 #g of pSVmyb. KNA was extracted as described (22) 48 h after transfectionexcept in lanes 3 and 4 (RNA extracted 24 and 36 h after transfection, respectively).To exclude amplification from genomic DNA, RT-PCR reactions were done in the absence of reversetranscriptase (-). In controls, reactions were also performed in the absence of RNA. Endogenous ~-actin mRNA levels Transactivation of CD34 L-CAT and CD34 LM-CAT was were also measured to ensure that a similar amount of RNA was analyzed abolished by the wild-type competitor (Fig. 2 C, bars 6 and for CD34 mP,NA expression. (B) Expression of CD34 mRNA in TK- 8) but was unaffected by the mutated oligomer (Fig. 2 C, ts13 cells constitutively expressing c-myb. (Left) levels of c-myb protein bars 10 and 12), further demonstrating that the transactivation determined by Western blot analysis. (Lane 1) untransfected TK-ts13 cells; of the CD34 promoter depends directly on c-rnyb expression (lane 2) SVmybTK-ts3cells; (lane 3) control HL-60 cells. (Right) CD34 mRNA levels determined by RT-PCR in untransfected TK-ts13 (lane 1) and interaction with Myb binding sites. The 36-bp competitor and in SV-myb TK-ts13 cells (lane 2) in the presence (+) or absence (-) containing two Myb binding sites was not toxic, as levels of reverse transcriptase; (lane 3) P,T-PCR reaction in the absence of RNA of CAT activity were identical in TK-ts13 cells transfected in the reaction mixture.

1025 Melotti et al. Brief Definitive Report A ,6 /, i i i rr UJ i m # .a Z oS i --t oW w

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Figure 4. CD34 mRNA levelsin T98G human glioblastoma cells constitutively expressing c-myb. (.4) Levels of c-myb protein were determined ".,t 0r ...... by Western blot analysis in untransfected T98G cells (lane I), in SV-myb- j. "..~ transfected T98G cells (lane 2), in SV-myb T98G cells after methotrexate illl~'~ ~ I , , ,lllll~ J I I'.~ selection (SVmyb M T98G) (lane 3), and in the control MEL cells (lane 4). (B) CD34 mRNA levels were determined by RT-PCR in the presence I0-~ 10o 101 102 (+) or absence (-) of reverse transcriptase. Each lane is representative of FLUORESCENCE INTENSITY, Ioglo three independent experiments with similar results. (Lane 1) negative con- Figure 5. Cell surface expression of CD34 in T98G constitutively ex- trol, HL-60 cells; (lane 2) T98G; cells, (lane 3) SV-myb T98G cells; (lane pressing c-myb. (A) KG-la cells: .... , negative control; -, anti-CD34. (B) 4) SV-myb T98G cells after methotrexate selection; (lane 5) positive con- -, T98G; anti-CD34; - - -, SVmybT98G; anti-CD34, ...-, SVmybMT98G; trol, KG-la cells (only one-fifth of the reaction was used); (lane 6) RT- anti-CD34, .-., negative control analysis, attached for the three cell lines, PCR reaction in the absence of RNA in the reaction mixture. is reported, x-axis, number of cells; y-axis, fluorescenceintensity, log scale. The analysis was repeated twice with identical results. of the c-rnyb transcription unit (Fig. 4 A, lane 3), but not in parental T98G cells (Fig. 4 A, lane 1). In SVmybM T98G tion. In contrast, expression of the CD34 stem cell marker cells, levels of c-myb protein expression were at least seven- declines during late stages of myeloid differentiation and matu- fold greater than in SVmyb T98G cells, as revealed by den- ration. Our data suggest that CD34 represents a primary target sitometric scanning of the film (Fig. 4 A, compare lanes 3 of c-myb regulation during the earliest recognizable stages and 4). of hematopoietic progenitor cell commitment and differen- Expression of CD34 mRNA was not detected in control tiation. A temporal relationship in which c-myb expression HL-60 cells (Fig. 4 B, lane I) or in nontransfected T98G precedes that of CD34 in human stem and progenitor cells cells (Fig. 4 B, lane 2), but was found in SVmyb T98G (Fig. is difficult to establish because primitive CD34-positive cells 4 B, lane 3), and, at higher levels, in SVmybMT98G cells express c-myb, and CD34-negative stem cells, if they exist, (Fig. 4 B, lane 4). Phenotypic analysis performed to monitor cannot be identified yet. A functional and temporal relation- surface expression of the human CD34 antigen on T98G, ship between c-myb and CD34 expression might be explored SVmyb T98G and SVmybMT98G cells (Fig. 5 B) indicated during the differentiation of embryonic stem cells. In these expression of CD34 antigen on a small proportion (4.3 +_ cells a stage can be identified at which c-myb, but not CD34, 1.2%) of T98G cells. Low density CD34 expression was de- is expressed (26); this observation may support the involve- tected on SVmyb T98G cells, and most SVmybMT98G ex- ment of c-myb in regulating CD34 expression during em- pressed CD34 at a variable level, with average density ap- bryonal hematopoietic development. proximately half of that detected in KG-la myeloblastic Our data clearly indicate that c-myb is a transcriptional leukemia cells used as positive control (Fig. 5 A). factor involved in regulating CD34 expression, but do not exclude that others are, too. Binding sites for ets proteins are present in the CD34 5' flanking region, some in close Discussion proximity with Myb binding sites, and preliminary findings The role of c-myb in hematopoiesis likely derives from its suggest that c-myb and ets-2 act synergistically to transacti- transactivating function, but the search for the relevant target(s) vate the CD34 promoter. In addition, it is likely that c-myb remains elusive, since two hematopoietic-associated targets, expression is the only prerequisite for CD34 expression, as the MIM-1 and the lysozyme genes identified in avian cells, implied by the lack of CD34 expression in the HL-60 cells are expressed at a relatively late stage of myeloid differentia- promyelocytic leukemia cells that express c-myb at high levels.

1026 Role of c-myb in Hematopoietic Stem Cell Antigen CD34 Expression KG-la and HL-60 represent relatively close stages of myeloid to control CD34 expression in early progenitor cells. Finally, differentiation and express c-myb at high levels, and yet only the increased fraction of CD34-positive cells in acute leukemia KG-la cells express CD34. This suggests either positive regu- patients correlates with the enhanced clonogenic potential lation of CD34 expression by a c-myb partner in KG-la cells, of these cells and their high levels of c-myb expression. In or negative regulation in HL-60 cells by a suppressor of CD34 conjunction with such observations, our data on c-myb regu- expression. In this regard, a functional binding site for the lation of CD34 expression might provide insights into the myeloid-specific transactivator NF-M contiguous to a c-myb coupling of differentiation arrest and growth advantage in binding site appears to be necessary for c-myb regulation of leukemic cells. the MIM-1 promoter (27); a similar mechanism may operate

We wish to thank David Dicker for performing the FACS| analysis and Dr. Bice Perussia for critical reading of the manuscript. P. Melotti was supported by a fellowship of the Associazione Italiana Ricerca sul Cancro (AIRC). D.-H.

Ku was supported by training grant 1T32 CA-096078 from the National Institutes of Health (NIH). Downloaded from http://rupress.org/jem/article-pdf/179/3/1023/1104540/1023.pdf by guest on 02 October 2021 B. Calabretta is a scholar of the Leukemia Society of America. This work was supported in part by NIH (CA-46782) and American Cancer Society (CH455A and CH492) grants to B. Calabretta. Address correspondence to Dr. Bruno Calabretta, Department of Microbiology and Immunology, Jefferson Cancer Institute, Bluemle Life Sciences Building, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107. P. Melotti is on leave from the Department of Pediatrics, University of Verona, Verona, Italy 37134.

Received for publication 1 November 1993 and in revised form I December 1993.

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1028 Role of c-myb in Hematopoietic Stem Cell Antigen CD34 Expression